Montmorillonite K-10 mediated green synthesis of cyano pyridines: Their evaluation as potential inhibitors of PDE4

Article abstract of DOI:10.1016/j.ejmech.2011.12.024

An efficient and green synthesis of functionalized cyano pyridines has been achieved via montmorillonite K-10 mediated multi-component reaction in a chemo- and regioselective manner. The four-component reaction of β-keto ester, arylaldehyde, malononitrile

Full text of DOI:10.1016/j.ejmech.2011.12.024

European Journal of Medicinal Chemistry 48 (2012) 265e274
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Montmorillonite K-10 mediated green synthesis of cyano pyridines: Their
evaluation as potential inhibitors of PDE4
,
,
T. Ram Reddy a b, G. Rajeshwar Reddya, L. Srinivasula Reddy a b, Subbarao Jammula a, Y. Lingappa b,
Ravikumar Kapavarapu c, Chandana Lakshmi T. Meda c, Kishore V.L. Parsa c, Manojit Pal c
,
*
a Custom Pharmaceutical Services, Dr. Reddy’s Laboratories Limited, Bollaram Road Miyapur, Hyderabad 500 049, India
b Department of chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
c Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and green synthesis of functionalized cyano pyridines has been achieved via montmorillonite
K-10 mediated multi-component reaction in a chemo- and regioselective manner. The four-component
Received 12 October 2011
Received in revised form
12 December 2011
Accepted 15 December 2011
Available online 21 December 2011
reaction of
b-keto ester, arylaldehyde, malononitrile and an alcohol provided a variety of pyridine
derivatives and montmorillonite K-10 was found to be a reusable catalyst. The potential of this opera-
tionally simple methodology has been demonstrated in further structure elaboration of a compound
synthesized via CeC bond forming reactions under Suzuki, Sonogashira and Heck conditions. Some of the
cyano pyridines synthesized showed PDE4B inhibitory properties in vitro and good interactions with
PDE4B protein in silico suggesting cyano pyridine scaffold as a potential template for the discovery of
novel PDE4 inhibitors.
Keywords:
Pyridine
Montmorillonite K-10
Ó 2011 Elsevier Masson SAS. All rights reserved.
b
-keto ester
Aldehyde
Malononitrile
PDE4
1. Introduction
The major strategies used for the synthesis of pyridine deriva-
tives include (i) modification of the pre-formed pyridine ring or (ii)
The prevalence of pyridines in nature (e.g., in the coenzyme
vitamin B6 family and in numerous alkaloids) and their central role
as versatile building blocks in the synthesis of natural products as
well as biologically active compounds has led to a continued interest
in the practical synthesis of pyridine derivatives [1]. The pyridine
nucleus has also been found to be integral part of several potent
inhibitors of phosphodiesterase 4 (PDE4) e.g. piclamilast, roflumilast
etc (Fig.1) that are known tobe beneficial for the potential treatment
of asthma and chronic obstructive pulmonary disease (COPD) [2a].
While roflumilast (DaxasÒ, Nycomed) [2b] has been launched in
Europe recently, due to the adverse side effects (e.g. nausea, emesis
and cardiovascular effects) shown by other inhibitors the search for
new chemical class for the evaluation of selective PDE4 inhibitory
properties with improved therapeutic indexes is desirable. In our
effort to identify novel inhibitors of PDE4 we became interested to
evaluate various 4-aryl substituted cyano pyridine derivatives (A,
Fig. 1) for their PDE4 inhibitory potential in vitro.
creation of the pyridine ring from suitably substituted open chain
precursor. Since multi-component reactions (MCRs) have emerged
as powerful tools in organic, combinatorial, and medicinal chem-
istry [3] particularly for easy access to diversified arrays of, e.g.,
valuable heterocyclic scaffolds their uses has also been reported in
the straightforward synthesis of pyridines. All these approaches
generally are based on carbonyl condensation chemistry and
frequently involve the use of aldehydes along with other suitable
reactants and reagents. Thus,
a one-pot synthesis of 3,5-
dicarbonitrile-pyridines has been reported by a MCR of an alde-
hyde, malononitrile, and a thiol in the presence of a base catalyst
such as piperidine, DABCO or triethylamine [4]. Similar synthesis of
pyridines was performed by using (i) a basic ionic liquid as both
catalyst and reaction medium [5], (ii) a range of Lewis acids [6] (e.g.
ZnCl2, AlCl3 and FeCl3), (iii) InCl3 [7], (iii) NaOH [8] and (iv) p-tol-
uenesulfonic acid [9]. The use of microwave irradiation to the MCR
preparation of pyridines has also been investigated [10]. While
useful for the preparation of various pyridine derivatives many of
these methods require the use of environmentally harmful cata-
lysts or reagents or longer reaction time. Thus simple and greener
synthetic methods for the construction of functionalized pyridine
* Corresponding author. Tel.: þ91 40 6657 1500; fax: þ91 40 6657 1581.
E-mail addresses: manojitpal@rediffmail.com, palmanojit@yahoo.co.uk (M. Pal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.024

266
T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
Initially, the MCR of 4-bromo benzaldehyde (1a), malononitrile
OCHF2
OMe
Y
(2), ethyl acetoacetate (3a) and ethanol (4a) in the presence or
absence of a solvent was examined under microwave irradiation
conditions (Table 1). A number of Lewis acids such as ZnCl2, AlCl3
and FeCl3 was examined but was found to be less effective as the
expected product 5a was isolated in 30e50% yield (entries 1e3,
Table 1). All these reactions were carried out for 10 min. To develop
an environmentally process we then examined the use of mont-
morillonite K-10 (entries 4e6, Table 1) and the reaction was carried
out for 4, 8 and 10 min separately. While the reaction proceeded
well in all these cases the best result however was obtained when
duration was 10 min (entry 6, Table 1). Spectral analysis of the
isolated product 5a suggested that ethanol played the role of
reactant as well as solvent in all these cases. However, the use of
additional aprotic solvents such as dimethylformamide (DMF) and
N-methyl pyrrolidone (NMP) decreased the product yield (entries 7
and 8, Table 1) indicating the use of ethanol was optimum. Notably,
all these reactions required excess amount of montmorillonite K-10
(200% w/w with respect to the aldehyde used) as the decreased
amount of catalyst (e.g. 75 or 150% w/w) decreased the product
yield. To test the recyclability of the catalyst, montmorillonite K-10
recovered by simple filtration was reused and the MCR afforded 5a
without affecting the yield significantly (entry 6, Table 1). While the
use of a similar methodology i.e. montmorillonite K-10 in combi-
nation with Pd/C under microwave irradiation towards the three
component synthesis of pyridine has been reported earlier [15] to
the best of our knowledge preparation of cyano pyridine 5a or its
analogues using this methodology has never been explored.
Having the optimized reaction conditions in hand we then
tested the generality and scope of this green methodology. Thus
reactants bearing varied substituted groups e.g. Ar, R1 and R2 were
employed under the optimized reaction conditions established
(Table 2). The reaction proceeded smoothly in all these cases to
afford a variety of cyano pyridines in good to excellent yields. Aryl
aldehydes 1 bearing mono (entries 1e6, 9, 14, 16, 17, Table 2), di
(entries 7,8, 18, Table 2) and tri substituted aromatic ring (entries 10
O
O
CN
X
HN
O
Cl
HN
O
Cl
Cl
Cl
N
X, Y = various
substituents
N
N
Piclamilast
roflumilast
A
Fig. 1. Pyridine based known PDE4 inhibitors and design of new inhibitors (A).
ring are still desirable. In continuation of our interest in pyridine
derivatives [11] of potential pharmacological interest [12] we now
wish to report a one-pot synthesis of substituted cyano pyridines
via four-component reaction of b-keto ester, arylaldehydes, malo-
nonitrile and an alcohol in the presence of montmorillonite K-10 as
a green and reusable catalyst (Scheme 1).
2. Results and discussion
2.1. Chemistry
Based on earlier reports on montmorillonite K-10 catalysed
Knoevenagel reaction [13] and Michael addition of 1,3-dicarbonyl
compounds [14] we envisioned that a Knoevenagel adduct (E-1)
generated in situ from an aldehyde and malononitrile may
undergoes Michael addition with a 1,3-dicarbonyl compound 3
(Scheme 2). The tautomeric form (E-2) of the intermediate thus
generated may reacts with an alcohol to generate an enamine (E-3)
which via intramolecular cyclization (Hantzsch pathway) and
subsequent aerial oxidation may provide a cyano pyridine deriva-
tive 5.
Ar
R2OH (4)
Montmoriilonite K10
R1O2C
CN
O
O
CN
ArCHO
+
+
OR1
N
OR2
CN
2
MW
1
3
5
Scheme 1. Green synthesis of substituted cyano pyridines through multi-component reaction.
OR1 Ar
CN
CN
OR1
OR1
NC
O
O
O
O
Ar
O
O
Ar
2
4
R OH ( )
E-1
CN
CN
C
3
HN
E-2
OR1
Ar
R1O2C
CN
OR2
O
Ar
[O]
5
O
CN
OR2
Hantzsch
pathway
N
H
C
H2N
E-3
Scheme 2. Proposed pathway for the montmorillonite K-10 mediated synthesis of substituted cyano pyridines.

T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
267
Table 1
Effect of reaction conditions on the multi-component reactions of 1a, 2, 3a and 4a.a
C6H4Br-p
4a
EtOH (
)
EtO2C
CN
O
O
CN
Catalyst
OHC
Br +
+
OEt
N
OEt
CN
2
MW
1a
3a
5a
Entry
Catalyst
Amountb
Time (min)
% yieldc
1.
2.
3.
4.
5.
6.
7.
8.
ZnCl2
AlCl3
FeCl3
2.0 equiv
2.0 equiv
2.0 equiv
200% w/w
200% w/w
200% w/w
200% w/w
200% w/w
10
10
10
4
30
40
50
50
Montmorillonite K-10
Montmorillonite K-10
Montmorillonite K-10
Montmorillonite K-10
Montmorillonite K-10
8
75
10
10
10
90 (88)d
55e
50f
a
All the reactions were carried out using 1a (200 mg, 2.0 mmol), 2 (71 mg, 2.0 mmol), 3 (140 mg, 2.0 mmol), a catalyst and ethanol 4a (5 mL) in a vessel that was sealed with
a pressure cap and irradiated in a CEM Explorer microwave at 120 ꢁC (300 W).
b
With respect to the aldehyde 1a used.
Isolated yield.
Recovered montmorillonite K-10 was used.
1.1 equiv of EtOH along with DMF (5 mL) was used.
c
d
e
f
1.1 equiv of EtOH along with NMP (5 mL) was used.
and 15, Table 2) or simple phenyl ring (entries 11e13, Table 2) were
employed and found to be effective in the present MCR.
amenable for further functionalization to generate diversity based
library of molecules.
To demonstrate the potential of this cyano pyridine synthesis
further structure elaboration of a compounds synthesized was
carried out through transition-metal mediated CeC bond forming
reactions. Thus compound 5b was derivatized via Sonogashira,
Suzuki and Heck reaction to afford compound 6, 7 and 8 (Scheme 3).
Mechanistically, the present one-pot MCR seems to proceed via
the pathway shown in Scheme 2. Thus four bonds were formed in
a single pot operation in highly chemo- and regioselective manner
to afford the functionalized cyano pyridines 5 under environmen-
tally friendly conditions. Many of these cyano pyridines are
2.2. Pharmacology
The selective inhibition of PDE4A and/or PDE4B without
affecting the other isoforms (thought to be responsible for unde-
sired side effects such as nausea and emesis) is the emerging
strategy to develop a safer drug [2]. On the other hand nicotinamide
derivatives have been reported as inhibitors of PDE4 [16]. Due to
our interest in PDE4 inhibitors [17] we therefore evaluated some of
the pyridines synthesized initially for their PDE4B inhibitory
potential in vitro at 30 mM using PDE4B enzyme assay [18] (Table 3).
Rolipram [19] was used as a reference compound in this assay. The
nature of substituent(s) present on the C-4 aryl moiety seemed to
have significant influence on PDE4 activities of the corresponding
compound. For example, a strong electron donating group e.g. OMe
at the para position of the C-4 benzene ring (entries 3 and 4,
Table 3) was found to be better than other milder donating groups
e.g. F or Br (entries 1,2, 5 and 6, Table 3). The di or tri substitution on
the C-4 benzene ring decreased the PDE4B inhibitory activities.
In order to understand the nature of interactions of these
molecules with PDE4B docking studies were carried out using the
compounds 5a (Fig. 2) and 5d (Fig. 3). The dock scores i.e. ꢀ17.08
and ꢀ25.47 kcal/mol obtained for 5a and 5d respectively suggests
that these molecules bind well with PDE4B. H-bonding interaction
was observed in the case of 5a involving the eCN group and His234
residue of PDE4B in addition to arene-cation interaction with the
same His234. Notably, the H-bonding interaction observed in the
case of 5d involved the eOMe group and Glu443 residue of PDE4B
in addition to the arene-arene interaction with Phe446 residue.
Table 2
Montmorillonite K-10 mediated synthesis of functionalized cyano pyridines through
MCR (Scheme 1).a
Entry
1; Ar]
3; R1] 4; R2] Products (5) Yieldb (%)
1.
2.
3.
4.
5.
6.
7.
8.
9.
1a; 4-BrC6H4
1a
1b; 4-MeOC6H4
1b
1c; 4-FC6H4
1c
1d; 2,4-di-FC6H3
1d
3a; Et
4a; Et
5a
90
85
72
75
81
85
87
79
81
71
82
73
79
76
75
89
91
88
3b; Me 4b; Me 5b
3b
3a
3a
3b
3b
3a
3b
4b
4a
4a
4b
4b
4a
4b
4b
4b
4a
4b
4a
4a
4b
4b
4b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
1e; 4-CF3C6H4
10. 1f; 2,6-di-F-4-BrC6H2 3b
11. 1g; C6H5
12. 1g
13. 1g
14. 1e
15. 1f
16. 1c
17. 1a
18. 1d
3b
3a
3a
3a
3a
3a
3a
3a
3. Conclusions
a
All the reactions were carried out using 1 (2.0 mmol), 2 (2.0 mmol), b-keto ester
3 (2.0 mmol), montmorillonite K-10 (400 mg, 2.0 times with respect to 1) and an
alcohol 4 (5 mL) in a vessel that was sealed with a pressure cap and irradiated in
a CEM Explorer microwave at 120 ꢁC (300 W).
In conclusion, a green and one-pot four-component synthesis of
functionalized cyano pyridines from readily available starting mate-
rials has been developed using montmorillonite K-10 as an efficient
b
Isolated yield.

268
T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
CH2(CH2)4CH3
C6H4OMe-p
p-MeOC6H4B(OH)2
Pd(PPh3)Cl2
n-Octyne
Pd(PPh3)Cl2
O
5b
o
CN
DMF/Et3N
K2CO3, 70-80 C
O
MeO
110-120 °C
CN
Methyl acrylate
Pd(PPh3)Cl2
DMF/Et3N
MeO
N
OMe
6
N
OMe
7
90-100 °C
CH=CHCO2Me
O
CN
MeO
N
OMe
8
Scheme 3. Suzuki, Sonogashira and Heck reaction of compound 5b.
and reusable catalyst. This operationally simple methodology
provided a variety of cyano pyridines in a chemo- and regioselective
manner. The potential of this methodology has been demonstrated in
further structure elaboration of a compound synthesized. Some of the
cyano pyridines synthesized showed PDE4B inhibitory properties
in vitro and good interactions with PDE4B protein in silico. The cyano
pyridine template presented here therefore has potential for the
discoveryof novelPDE4inhibitors andthemethodologythereforewill
find applications in the synthesis of pyridine based library of mole-
cules of potential pharmacological interest.
(400 mg, 200% w/w with respect to the aldehyde used). The reaction
vessel was sealed with a pressure cap and irradiated in a CEM
Explorer microwavefor 10 min at 120 ꢁC (300 W) (the progressof the
reaction was monitored by TLC). After completion, the reaction
mixture was cooled to room temperature, filtered through a celite
bedandthe bedwaswashedwithdichloromethane (2 ꢂ 5.0 mL). The
filtrates were collected, combined and concentrated under reduced
pressure. The residue was purified bycolumn chromatography using
ethyl acetate/hexanes (0e15%) to give the desired product.
4.1.3. Ethyl 4-(4-bromophenyl)-5-cyano-6-ethoxy-2-
methylnicotinate (5a)
4. Experimental section
Light yellow solid (379 mg, 0.90 mmol, 90%); mp 136e137 ꢁC; IR
(KBr) 2964, 2229, 1725, 1586, 1548, 1379, 1337, 1274, 1158,
4.1. Chemistry
1073 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.92 (t, J ¼ 7.2 Hz, 3H), 1.43
(t, J ¼ 7.6 Hz, 3H), 2.57 (s, 3H), 4.0 (q, J ¼ 7.0 Hz, 2H), 4.53 (q,
4.1.1. General methods
J ¼ 7.6 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz, 2H); 13C
Unless stated otherwise, reactions were monitored by thin layer
chromatography (TLC) on silica gel plates (60 F254), visualizing with
ultraviolet light or iodine spray. Column chromatography was per-
formed on silica gel (60e120 mesh) using distilled petroleum ether
and ethyl acetate.1H and 13C NMR spectra were determined in CDCl3/
DMSO-d6 solution using 400 and 100 MHz spectrometers, respec-
NMR (CDCl3, 100 MHz) d 13.5,14.3, 23.4, 61.6, 63.8, 93.9,114.2,122.6,
124.0, 129.6 (2C), 131.8 (2C), 134.0, 153.7, 159.5, 163.5, 166.8; HRMS:
m/z calcd for C18H17BrN2O3 (M þ 1) 389.0423; found 389.0507.
4.1.4. Methyl 4-(4-bromophenyl)-5-cyano-6-methoxy-2-
methylnicotinate (5b)
tively. Proton chemical shifts (
d) are relative to tetramethylsilane
Light yellow solid (334 mg, 0.85 mmol, 85%); mp 112e113 ꢁC; IR
(KBr) 2940, 2226, 1732, 1591, 1551, 1473, 1378, 1274, 1152,
(TMS,
d
¼ 0.0) as internal standard and expressed in parts per million.
Spin multiplicities are given as s (singlet), d (doublet), t (triplet), and
m (multiplet) as well as b (broad). Coupling constants (J) are given in
hertz. Infrared spectrawere recorded on a FTIR spectrometer. Melting
points were determined by using a Buchi melting point B-540
apparatus. MS spectra were obtained on a mass spectrometer. HRMS
wasdeterminedusing watersLCT premier XETOFARE-047 apparatus.
1081 cmꢀ1 1H NMR (CDCl3, 400 MHz)
; d 2.50 (s, 3H), 3.48 (s, 3H),
4.02 (s, 3H), 7.16 (d, J ¼ 8.8 Hz, 2H), 7.52 (d, J ¼ 8.4 Hz, 2H); 13C NMR
(CDCl3, 100 MHz)
d 23.5, 52.4, 54.9, 93.9, 114.1, 122.7, 124.3, 129.5,
131.9 (2C), 132.1, 133.7, 153.7, 159.5, 163.3, 167.3; HRMS: m/z calcd
for C16H13BrN2O3 (M þ 1) 361.0110; found 361.0174.
4.1.5. Methyl 5-cyano-6-methoxy-4-(4-methoxyphenyl)-2-
methylnicotinate (5c)
4.1.2. Typical procedure for the synthesis of substituted cyano
pyridine 5a
A mixture of ethyl acetoacetate (140 mg, 2.0 mmol), 4-bromo
benzaldehyde (200 mg, 2.0 mmol), and malononitrile (71 mg,
2.0 mmol) in ethanol (5 mL) was added montmorillonite K-10
Off white solid (330 mg, 0.72 mmol, 72%); mp 137e138 ꢁC; IR
(KBr) 2940, 2225, 1732, 1579, 1557, 1470, 1388, 1297, 1152,
1079 cmꢀ1 1H NMR (CDCl3, 400 MHz)
; d 2.56 (s, 3H), 3.57 (s, 3H),

T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
Table 3 (continued )
269
Table 3
Inhibition of PDE4B by compound 5 at 30 mM.
Entry
Compounds
Average %
inhibition
SD
Entry
Compounds
Average %
inhibition
SD
7.
21.46
1.34
1.
43.72
3.92
5g
5a
2.
44.80
2.17
8.
20.43
2.31
5b
5h
3.
64.10
2.15
9.
23.47
1.63
5c
5i
4.
65.90
3.20
10.
22.44
2.75
5d
5j
5.
42.34
1.29
11.
12.
38.42
90.12
1.56
4.35
5e
5k
Rolipram
SD ¼ standard deviation.
6.
43.21
3.22
3.81 (s, 3H), 4.08 (s, 3H), 6.82 (d, J ¼ 8.4 Hz, 2H), 7.08 (d, J ¼ 8.0 Hz,
2H); 13C NMR (CDCl3, 100 MHz)
23.3, 52.3, 54.9, 55.3, 93.9, 114.3,
d
115.6, 115.9, 118.4, 130.0, 130.1, 131.1, 154.6, 159.4, 159.5, 163.9, 167.6;
HRMS: m/z calcd for C17H16N2O4 (M þ 1)þ 313.1110; found 313.1181.
5f

270
T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
Fig. 2. Schematic representation of the interaction between 5a and PDE4B. Hydrophobic amino acids are coloured green, polar are purple. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Schematic representation of the interaction between 5d and PDE4B. Hydrophobic amino acids are coloured green, polar are purple. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
271
4.1.6. Ethyl 5-cyano-6-ethoxy-4-(4-methoxyphenyl)-2-
4.1.12. Methyl-4-(4-bromo-2,6-difluorophenyl)-5-cyano-6-
methylnicotinate (5d)
methoxy-2-methylnicotinate (5j)
Off white solid (375 mg, 0.75 mmol, 75%); mp 125e127 ꢁC; IR
(KBr) 2925, 2226, 1738, 1589, 1551, 1463, 1375, 1249, 1179,
Off white solid (256 mg, 0.71 mmol, 71%); mp 124e125 ꢁC; IR
(KBr) 2925, 2232, 1732, 1625, 1556, 1470, 1388, 1274, 1152,
1086 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.94 (t, J ¼ 7.2 Hz, 3H), 1.42
1071 cmꢀ1
;
1H NMR (CDCl3, 400 MHz)
d
2.68 (s, 3H), 3.66 (s, 3H),
(t, J ¼ 7.2 Hz, 3H), 2.57 (s, 3H), 3.79 (s, 3H), 4.01 (q, J ¼ 7.2 Hz, 2H),
4.12 (s, 3H), 7.21e7.26 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d
24.5,
4.62 (q, J ¼ 7.2 Hz 2H), 6.95 (d, J ¼ 8.4 Hz, 2H), 7.15 (d, J ¼ 8.4 Hz,
52.4, 55.0, 95.9, 113.4, 115.6, 122.1, 124.0, 124.1, 143.7, 157.7, 160.2,
160.3, 161.6, 163.4, 165.4; HRMS: m/z calcd for C16H11BrF2N2O3
(M þ 1) 396.9921; found 396.9982.
2H); 13C NMR (CDCl3, 100 MHz)
d 13.5, 14.2, 24.5, 55.2, 61.6, 63.8,
93.8, 114.2, 115.6, 115.9, 118.2, 130.0, 130.1, 131.0, 154.7, 159.1, 159.5,
164.0, 167.7; HRMS: m/z calcd for C19H20N2O4 (M þ 1) 341.1423;
found 341.1497.
4.1.13. Methyl 5-cyano-6-methoxy-2-methyl-4-phenylnicotinate
(5k)
4.1.7. Ethyl 5-cyano-6-ethoxy-4-(4-fluorophenyl)-2-methylnicotinate
(5e)
Light yellow solid (436 mg, 0.82 mmol, 82%); mp 122e123 ꢁC; IR
(KBr) 2949, 2226, 1732, 1558, 1496, 1391, 1276, 1152, 1078 cmꢀ1; 1H
Light yellow solid (428 mg, 0.81 mmol, 81%); mp 127e129 ꢁC; IR
(KBr) 2948, 2224, 1739, 1596, 1562, 1472, 1379, 1268, 1152,
NMR (CDCl3, 400 MHz)
7.31e7.39 (m, 2H), 7.41e7.46 (m, 3H); 13C NMR (CDCl3, 100 MHz)
23.4, 52.2, 54.8, 94.0, 114.3, 122.9, 127.8 (2C), 128.6 (2C), 129.6,
d 2.58 (s, 3H), 3.51 (s, 3H), 4.10 (s, 3H),
1077 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.93 (t, J ¼ 7.2 Hz, 3H), 1.43
d
(t, J ¼ 6.8 Hz, 3H), 2.56 (s, 3H), 4.01 (q, J ¼ 7.0 Hz, 2H), 4.53
134.9, 155.0, 159.1, 163.9, 167.6; HRMS: m/z calcd for C16H14N2O3
(M þ 1) 283.1004; found 283.1073.
(q, J ¼ 6.8 Hz, 2H), 7.14 (d, J ¼ 7.2 Hz, 2H), 7.52 (d, J ¼ 6.8 Hz, 2H); 13C
NMR (CDCl3, 100 MHz) d 13.5, 14.3, 23.4, 61.6, 63.7, 94.0, 114.3, 115.7,
115.9, 122.8, 130.0, 130.1, 130.2, 153.8, 159.3, 162.1, 164.6, 167.0;
HRMS: m/z calcd for C18H17FN2O3 (M þ 1) 329.1223; found
329.1290.
4.1.14. Ethyl 5-cyano-6-ethoxy-2-methyl-4-phenylnicotinate (5l)
Light yellow solid (427 mg, 0.73 mmol, 73%); mp 137e139 ꢁC; IR
(KBr) 2980, 2227, 1723, 1557, 1446, 1383, 1340, 1272, 1154,
1077 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.98 (t, J ¼ 7.2 Hz, 3H), 1.43
4.1.8. Methyl 5-cyano-4-(4-fluorophenyl)-6-methoxy-2-methylnico-
tinate (5f)
(t, J ¼ 7.2 Hz, 3H), 2.57 (s, 3H), 3.95 (q, J ¼ 7.2 Hz, 2H), 4.53 (q, J ¼ 7.2
and 7.2 Hz, 2H), 7.32e7.37 (m, 2H), 7.44e7.45 (m, 3H); 13C NMR
Off white solid (411 mg, 0.85 mmol, 85%); mp 131e133 ꢁC; IR
(KBr) 2948, 2224, 1738, 1596, 1561, 1472, 1379, 1268, 1165,
(CDCl3, 100 MHz) d 13.4, 14.3, 23.3, 61.4, 63.6, 94.0, 114.3, 122.8,
128.0, 128.5, 128.7, 128.9, 129.4, 135.1, 154.9, 159.1, 163.5, 167.0;
HRMS: m/z calcd for C18H18N2O3 (M þ 1) 311.1317; found 311.1387.
1077 cmꢀ1
;
1H NMR (CDCl3, 400 MHz)
d
2.58 (s, 3H), 3.55 (s, 3H),
4.10 (s, 3H), 7.16 (d, J ¼ 8.4 Hz, 2H), 7.35 (d, J ¼ 8.4 Hz, 2H); 13C NMR
(CDCl3, 100 MHz)
d
23.4, 52.4, 54.9, 94.1, 114.2, 115.8, 116.0, 122.9,
4.1.15. Ethyl 5-cyano-6-methoxy-2-methyl-4-phenylnicotinate (5m)
White solid (441 mg, 0.79 mmol, 79%); mp 117e118 ꢁC; IR (KBr)
2986, 2226, 1726, 1562, 1472, 1379, 1354, 1272, 1154, 1076 cmꢀ1; 1H
129.9, 130.0, 130.9, 153.9, 159.3, 163.9, 164.6, 167.5; HRMS: m/z calcd
for C16H13FN2O3 (M þ 1) 301.0910; found 301.0972.
NMR (CDCl3, 400 MHz)
J ¼ 7.2 Hz, 2H), 4.10 (s, 3H), 7.31e7.38 (m, 2H), 7.42e7.45 (m, 3H);
13C NMR (CDCl3, 100 MHz)
13.4, 23.3, 54.8, 61.5, 93.9, 114.4, 123.1,
d
0.89 (t, J ¼ 7.2 Hz, 3H), 2.60 (s, 3H), 3.94 (q,
4.1.9. Methyl 5-cyano-4-(2,4-difluorophenyl)-6-methoxy-2-methyl-
nicotinate (5g)
d
Light yellow solid (390 mg, 0.87 mmol, 87%); mp 151e152 ꢁC; IR
(KBr) 2956, 2227, 1736, 1597, 1563, 1474, 1381, 1273, 1150,
127.9 (2C), 128.5 (2C), 129.5, 135.0, 155.0, 159.2, 163.7, 167.0; HRMS:
m/z calcd for C17H16N2O3 (M þ 1) 297.1161; found 297.1234.
1075 cmꢀ1
;
1H NMR (CDCl3, 400 MHz)
d
2.62 (s, 3H), 3.59 (s, 3H),
4.11 (s, 3H), 6.93 (m, 2H), 7.21 (m, 1H); 13C NMR (CDCl3, 100 MHz)
23.9, 52.3, 54.9, 95.3, 104.5, 111.7, 113.7, 119.1, 122.8, 130.8, 158.0,
4.1.16. Ethyl 5-cyano-6-ethoxy-2-methyl-4-(4-(trifluoromethyl)
phenyl)nicotinate (5n)
d
160.4, 162.5, 163.7, 165.0, 166.7; HRMS: m/z calcd for C16H12F2N2O3
(M þ 1) 319.0816; found 319.0885.
White solid (330 mg, 0.76 mmol, 76%); mp 126e128 ꢁC; IR (KBr)
2993, 2232, 1723, 1555, 1445, 1382, 1338, 1275, 1163, 1067 cmꢀ1; 1H
NMR (CDCl3, 400 MHz)
d
0.86 (t, J ¼ 7.2 Hz, 3H), 1.44 (t, J ¼ 7.2 Hz,
4.1.10. Ethyl 5-cyano-4-(2,4-difluorophenyl)-6-ethoxy-2-
methylnicotinate (5h)
3H), 2.60 (s, 3H), 3.96 (q, J ¼ 7.2 Hz, 2H), 4.55 (q, J ¼ 7.2 Hz, 2H), 7.47
(d, J ¼ 8.4 Hz, 2H), 7.72 (d, J ¼ 8.4 Hz, 2H); 13C NMR (CDCl3,
Light yellow solid (385 mg, 0.79 mmol, 79%); mp 145e146 ꢁC; IR
(KBr) 2979, 2226, 1731, 1620, 1559, 1477, 1343, 1270, 1177,
100 MHz): d 13.3, 14.2, 23.5, 61.6, 63.9, 93.9, 113.9, 122.4, 125.0,
125.5 (2C), 128.5 (2C), 131.4, 138.8, 153.4, 159.9, 163.5, 166.5; HRMS:
m/z calcd for C19H17F3N2O3 (M þ 1) 379.1191; found 379.1256.
1100 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.98 (t, J ¼ 7.2 Hz, 3H), 1.44
(t, J ¼ 7.6 Hz, 3H), 2.61 (s, 3H), 4.03 (q, J ¼ 7.0 Hz, 2H), 4.54
(q, J ¼ 7.4 Hz, 2H), 6.93 (m, 2H), 7.22 (m, 1H); 13C NMR (CDCl3,
4.1.17. Ethyl 4-(4-bromo-2,6-difluorophenyl)-5-cyano-6-ethoxy-2-
methylnicotinate (5o)
100 MHz) d 13.5, 14.3, 23.9, 61.5, 63.8, 94.0, 104.5, 111.8, 113.8, 119.2,
122.7, 130.9, 158.0, 160.5, 162.4, 163.3, 164.9, 166.2; HRMS: m/z calcd
for C18H16F2N2O3 (M þ 1) 347.1129; found 347.1207.
Light yellow solid (289 mg, 0.75 mmol, 75%); mp 132e133 ꢁC; IR
(KBr) 2981, 2231, 1731, 1624, 1563, 1490, 1386, 1270, 1163,
1073 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
1.03 (t, J ¼ 7.6 Hz, 3H), 1.44
4.1.11. Methyl 5-cyano-6-methoxy-2-methyl-4-(4-(trifluoromethyl)
phenyl)nicotinate (5i)
(t, J ¼ 7.2 Hz, 3H), 2.67 (s, 3H), 4.09 (q, J ¼ 7.2 Hz, 2H), 4.55
(q, J ¼ 7.4 Hz, 2H), 7.21e7.26 (m, 2H); 13C NMR (CDCl3, 100 MHz)
Light yellow solid (326 mg, 0.81 mmol, 81%); mp 112e113 ꢁC;
IR (KBr) 2954, 2230, 1731, 1620, 1556, 1472, 1379, 1276, 1154,
d 13.5, 14.2, 24.5, 61.6, 64.0, 95.9, 113.3, 115.5, 122.1, 123.8, 123.9,
143.5, 157.6, 160.1, 160.2, 161.7, 163.6, 165.9; HRMS; m/z calcd for
C18H15BrF2N2O3 (M þ 1) 425.0234; found 425.0303.
1067 cmꢀ1 1H NMR (CDCl3, 400 MHz)
; d 2.60 (s, 3H), 3.53 (s, 3H),
4.12 (s, 3H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.72 (d, J ¼ 8.4 Hz, 2H); 13C
NMR (CDCl3, 100 MHz)
d
23.5, 52.3, 54.9, 93.9, 113.9, 122.3, 122.5,
4.1.18. Ethyl 5-cyano-4-(4-fluorophenyl)-6-methoxy-2-
methylnicotinate (5p)
125.0, 125.6, 128.4 (2C), 131.4, 138.5, 153.4, 159.9, 163.8, 167.0;
HRMS: m/z calcd for C17H13F3N2O3 (M þ 1) 351.0878; found
351.0945.
White solid (450 mg, 0.89 mmol, 89%); mp 110e111 ꢁC; IR (KBr)
2964, 2224, 1730, 1596, 1561, 1471, 1378, 1340, 1267, 1152,

272
T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
1076 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.94 (t, J ¼ 7.2 Hz, 3H), 2.58
compound as a light yellow solid (162 mg, 0.75 mmol, 75%); mp
(s, 3H), 4.00 (q, J ¼ 7.2 Hz, 2H), 4.10 (s, 3H), 7.13 (d, J ¼ 8.0 Hz, 2H),
153e154 ꢁC; IR (KBr) 2930, 2857, 2229, 1731, 1568, 1512, 1469, 1379,
7.32 (d, J ¼ 8.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz):
d
13.5, 23.3, 54.8,
1275, 1153, 1079 cmꢀ1 1H NMR (CDCl3, 400 MHz)
; d 0.91
61.6, 94.0, 114.2, 115.6, 115.8, 123.1, 130.0, 130.1, 130.9, 153.8, 159.3,
163.7 (2C), 166.9; HRMS: m/z calcd for C17H15FN2O3 (M þ 1)
315.1067; found 315.1134.
(t, J ¼ 6.8 Hz, 3H), 1.23e1.63 (m, 8H), 2.39 (t, J ¼ 6.8 Hz, 2H), 2.61
(s, 3H), 3.54 (s, 3H), 4.05 (s, 3H), 7.17 (d, J ¼ 8.4 Hz, 2H), 7.58
(d, J ¼ 8.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz)
d 14.0, 22.6, 23.4, 28.5,
29.6, 31.3, 31.8, 52.3, 54.9, 79.8, 92.6, 93.8, 114.2, 122.7, 124.2, 125.7,
127.8, 129.5, 131.9, 133.7, 154.4, 159.3, 163.9, 167.5; HRMS: m/z calcd
for C24H26N2O3 (M þ 1) 391.1943; found 391.1997.
4.1.19. Ethyl 4-(4-bromophenyl)-5-cyano-6-methoxy-2-
methylnicotinate (5q)
White solid (369 mg, 0.91 mmol, 91%); mp 125e127 ꢁC; IR (KBr)
2955, 2228, 1725, 1592, 1553, 1469, 1384, 1276, 1151, 1080 cmꢀ1; 1H
4.1.23. (E)-methyl 5-cyano-6-methoxy-4-(4-(3-methoxy-3-
NMR (CDCl3, 400 MHz)
d
0.95 (t, J ¼ 7.2 Hz, 3H), 2.59 (s, 3H), 4.00
oxoprop-1-en-1-yl)phenyl)-2-methylnicotinate (8)
(q, J ¼ 7.0 Hz, 2H), 4.10 (s, 3H), 7.22 (d, J ¼ 8.4 Hz, 2H), 7.59 (d,
A mixture of bromo pyridine 5b (200 mg, 0.55 mmol), methyl
acrylate (95 mg, 1.11 mmol), (PPh3)2PdCl2 (19.44 mg, 0.05 mmol),
Et3N (196 mg, 1.94 mmol) in DMF (5 mL) was stirred at room temp
for 15 min and then at 90 ꢁC for 6 h under nitrogen (progress of the
reaction was monitored by TLC). After completion, the reaction
mixture was cooled to room temp and filtered through celite bed.
The filtrate was extracted with ethylacetate (3 ꢂ 5.0 mL). The
organic layers were collected, combined, dried over anhydrous
Mg2SO4, and concentrated under vacuum. The residue was purified
by column chromatography using EtOAc/Hexane (0e20%) to give
the title compound as a light yellow solid (158 mg, 0.78 mmol,
78%); mp 135e137 ꢁC; IR (KBr) 2962, 2867, 2231, 1726, 1596, 1561,
J ¼ 8.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz)
d 13.5, 23.3, 54.8, 61.6,
93.8, 114.1, 122.8, 124.1, 129.6 (2C), 131.8 (2C), 133.8, 153.7, 159.5,
163.7, 166.7; HRMS: m/z calcd for C17H15BrN2O3 (M þ 1) 375.0266;
found 375.0331.
4.1.20. Ethyl 5-cyano-4-(2,4-difluorophenyl)-6-methoxy-2-
methylnicotinate (5r)
Light yellow solid (408 mg, 0.88 mmol, 88%); mp 147e148 ꢁC; IR
(KBr) 2956, 2228, 1736, 1596, 1563, 1473, 1380, 1269, 1168,
1075 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d
0.98 (t, J ¼ 7.2 Hz, 3H), 2.63
(s, 3H), 4.04 (q, J ¼ 7.2 Hz, 2H), 4.11 (s, 3H), 6.93e6.99 (m, 2H),
7.25e7.27 (m,1H); 13C NMR (CDCl3, 100 MHz)
13.5, 23.8, 54.9, 61.5,
d
1483, 1377, 1276, 1162, 1079 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d 2.50
95.3, 104.7, 111.8, 113.7, 119.1, 123.0, 130.8, 158.0, 160.3, 162.5, 163.5,
165.0, 166.1; HRMS: m/z calcd for C17H14F2N2O3 (M þ 1) 333.0972;
found 333.1044.
(s, 3H), 3.47 (s, 3H), 3.75 (s, 3H), 4.03 (s, 3H), 6.35 (d, J ¼ 16.0 Hz,1H),
7.17 (d, J ¼ 8.4 Hz, 2H), 7.54 (d, J ¼ 8.0 Hz, 2H), 7.61 (d, J ¼ 16.0 Hz,
1H); 13C NMR (CDCl3, 100 MHz)
d 23.5, 52.4, 54.9, 56.6, 94.0, 114.1,
115.3, 122.7, 124.3, 129.5 (2C), 131.9 (2C), 132.1, 133.7, 153.7, 159.5,
163.9, 165.0, 167.3; HRMS: m/z calcd for C20H18N2O5 (M þ 1)
367.1216; found 367.1255.
4.1.21. Preparation of methyl 5-cyano-6-methoxy-4-(4’-methoxy-
[1,1’-biphenyl]-4-yl)-2-methylnicotinate (6)
A mixture of bromo pyridine 5b (200 mg, 0.55 mmol), aryl
boronic acid (101 mg, 0.66 mmol), (PPh3)2PdCl2 (19.44 mg,
0.05 mmol,) and anhydrous K2CO3 (230 mg, 1.65 mmol) in dioxane
(5 mL) was stirred initially at room temp for 15 min and then at
70 ꢁC for 3 h under nitrogen (progress of the reaction was moni-
tored by TLC). After completion, the reaction mixture was cooled to
room temp and filtered through celite bed. The filtrate was
extracted with ethylacetate (3 ꢂ 5.0 mL). The organic layers were
collected, combined, dried over anhydrous Mg2SO4, and concen-
trated under vacuum. The residue was purified by column chro-
matography using EtOAc/Hexane (0e15%) to give the title
compound as a light yellow solid (178 mg, 0.83 mmol, 83%); mp
173e174 ꢁC; IR (KBr) 2952, 2223, 1724, 1610, 1567, 1470, 1380, 1279,
5. Pharmacology
5.1. Materials and methods
Cells and Reagents: HEK 293 and Sf9 cells were obtained from
ATCC (Washington D.C., USA). HEK 293 cells were cultured in
DMEM supplemented with 10% fetal bovine serum (Invitrogen Inc.,
San Diego, CA, USA). Sf9 cells were routinely maintained in Grace’s
supplemented medium (Invitrogen) with 10% FBS. RAW 264.7 cells
(murine macrophage cell line) were obtained from ATCC and
routinely cultured in RPMI 1640 medium with 10% fetal bovine
serum (Invitrogen Inc.). cAMP was purchased from SISCO Research
Laboratories (Mumbai, India). PDElight HTS cAMP phosphodies-
terase assay kit was procured from Lonza (Basel, Switzerland).
1148, 1079 cmꢀ1 1H NMR (CDCl3, 400 MHz)
; d 2.58 (s, 3H), 3.56
(s, 3H), 3.86 (s, 3H), 4.11 (s, 3H), 6.98 (d, J ¼ 2.0 Hz, 2H), 7.40
(d, J ¼ 8.4 Hz, 2H), 7.57 (d, J ¼ 2.0 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz, 2H); 13C
NMR (CDCl3, 100 MHz)
d
23.3, 52.3, 54.8, 55.2, 93.8, 114.2, 114.5,
5.2. PDE4B protein production and purification
122.9, 126.7 (2C), 128.1 (2C), 128.3 (2C), 132.3 (2C), 132.9, 141.9,
154.7, 159.1, 159.5, 163.9, 167.7; HRMS: m/z calcd for C23H20N2O4
(M þ 1) 389.1423; found 389.1496.
PDE4B cDNA was sub-cloned into pFAST Bac HTB vector (Invi-
trogen) and transformed into DH10Bac (Invitrogen) competent
cells. Recombinant bacmids were tested for integration by PCR
analysis. Sf9 cells were transfected with bacmid using Lipofect-
amine 2000 (Invitrogen) according to manufacturer’s instructions.
Subsequently, P3 viral titer was amplified, cells were infected and
48 h post infection cells were lysed in lysis buffer (50 mM TriseHCl
pH 8.5, 10 mM 2-Mercaptoethanol, 1% protease inhibitor cocktail
(Roche), 1% NP40). Recombinant His-tagged PDE4B protein was
purified as previously described elsewhere [18]. Briefly, lysate was
centrifuged at 10,000 rpm for 10 min at 4 ꢁC and supernatant was
collected. Supernatant was mixed with Ni-NTA resin (GE Life
Sciences) in a ratio of 4:1 (v/v) and equilibrated with binding buffer
(20 mM TriseHCl pH 8.0, 500 mM KCl, 5 mM imidazole, 10 mM 2-
mercaptoethanol and 10% glycerol) in a ratio of 2:1 (v/v) and
mixed gently on rotary shaker for 1 h at 4 ꢁC. After incubation,
4.1.22. Preparation of methyl 5-cyano-6-methoxy-2-methyl-4-
(4-(oct-1-yn-1-yl)phenyl)nicotinate (7)
A mixture of bromo pyridine 5b (200 mg, 0.55 mmol), n-octyne
(121 mg, 1.1 mmol), (PPh3)2PdCl2 (19.44 mg, 0.05 mmol), CuI
(52 mg, 0.27 mmol) and Et3N (168 mg, 1.66 mmol) in DMF (5 mL)
was stirred initially at room temp for 15 min and then at
110e120 ꢁC for 5 h under nitrogen reaction (progress of the reaction
was monitored by TLC). After completion, the reaction mixture was
cooled to room temp and filtered through celite bed. The filtrate
was extracted with ethylacetate (3 ꢂ 5.0 mL). The organic layers
were collected, combined, dried over anhydrous Mg2SO4, and
concentrated under vacuum. The residue was purified by column
chromatography using EtOAc/Hexane (0e10%) to give the title

T. Ram Reddy et al. / European Journal of Medicinal Chemistry 48 (2012) 265e274
273
lysate-Ni-NTA mixturewas centrifuged at 4500 rpm for 5 min at 4 ꢁC
and the supernatant was collected as the flow-through fraction.
Resin was washed twice with wash buffer (20 mM TriseHCl pH 8.5,
1 M KCl, 10 mM 2-Mercaptoethanol and 10% glycerol). Protein was
eluted sequentially twice using elution buffers (Buffer I: 20 mM
TriseHCl pH 8.5, 100 mM KCl, 250 mM imidazole, 10 mM 2-
mercaptoethanol, 10% glycerol, Buffer II: 20 mM TriseHCl pH 8.5,
100 mM KCl, 500 mM imidazole, 10 mM 2-mercaptoethanol, 10%
glycerol). Eluates were collected in four fractions and analyzed by
SDS-PAGE. Eluates containing PDE4B protein were pooled and
stored at ꢀ80 ꢁC in 50% glycerol until further use.
For all scoring functions, lower scores indicate more favorable
poses. The unit for all scoring functions is kcal/mol. The final energy
was calculated using the Generalized Born solvation model. Poses
for each ligand were scored based on complementarity with the
binding pocket.
The London dG scoring function estimates the free energy of
binding of the ligand from a given pose. The functional form is
a sum of terms:
X
X
X
D
G ¼ c þ Eflex
cHB HB
f
þ
cMfM
D
Di
hꢀbonds
mꢀlig
atoms i
where c represents the average gain/loss of rotational and trans-
lational entropy; Eflex is the energy due to the loss of flexibility of
the ligand (calculated from ligand topology only); fHB measures
geometric imperfections of hydrogen bonds and takes a value in
[0,1]; cHB is the energy of an ideal hydrogen bond; fM measures
geometric imperfections of metal ligations and takes a value in
[0,1]; cM is the energy of an ideal metal ligation; and Di is the
desolvation energy of atom i.
5.3. PDE4B enzymatic assay
The inhibition of PDE4B enzyme was measured using PDElight
HTS cAMP phosphodiesterase assay kit (Lonza) according to
manufacturer’s recommendations. Briefly, 10 ng of PDE4B enzyme
was pre-incubated either with DMSO (vehicle control) or
compound for 15 min before incubation with the substrate cAMP
To validate the Docking accuracy of the program used, the native
co-crystallized Roflumilast was docked back into its binding site of
PDE4B Protein.
(5 mM) for 1 h. The reaction was halted with stop solution followed
by incubation with detection reagent for 10 min in dark. Lumi-
nescence values (RLUs) were measured by a Multilabel plate reader
(Perkin Elmer 1420 Multilabel counter). The percentage of inhibi-
tion was calculated using the following formula:
Acknowledgments
The authors (TRR, GRR, LSR, and SJ) thank Dr. Vilas Dahanukar
and Dr. Amrendra Kumar Roy for support and the analytical group
of DRL for spectral data.
ðRLU of vehicle control ꢀ RLU of inhibitorÞ
%inhibition ¼
ꢂ 100
RLU of vehicle control
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.12.024.
6. Docking study
The molecular docking simulation was carried out using
Chemical Computing Group’s Molecular Operating Environment
(MOE) software 2008.10 Version, “DOCK” application Module. The
molecule 5a and 5d was docked in the PDE4B protein and their
respective docking scores and interactions were observed.
Procedure: The PDE4B receptor from the structure of PDE4B in
complex with Roflumilast (PDB code 1XMU) was used for docking
studies. The original 1XMU PDB file contains crystallized Zn and Mn
metal ions. The PDE4B Protein was retrieved from PDB and
protonated (addition of hydrogen atoms) with protonation 3D
application in MOE. Connolly Molecular surface was generated
around the ligand site of the protein. Gasteiger Partial charges were
added to the protein and finally energy minimized to relieve bad
crystallographic contacts. “Active site finder” function of the MOE
software was used to denote potential docking pockets within the
protein crystal structure. The molecule 5a and 5d was placed in the
active site pocket of the protein by the “Triangle Matcher” method,
which generated poses by aligning the ligand triplet of atoms with
the triplet of alpha spheres in cavities of tight atomic packing. Dock
scoring was performed using London dG method. The best 10 poses
of molecules were retained and scored. The preparation of ligands
for docking simulation involved energy minimization with Molec-
ular Mechanics Force-field MMFF94x (Merck Molecular Force Field
94x). Molecules were then subjected to conformational search in
MOE using the Conformations Stochastic search module to find the
lowest energy conformers.
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